Given the limited genetic coding capacity of HIV-1, it is reasonable to expect that the virus must interact with an extensive battery of cellular factors to complete its passage through the cell. Indeed, it is remarkable that the viral genome, comprising only about 0.0003% of the entire genetic capacity of the cell, commandeers the cellular environment to its own advantage. However, to date and despite being easily the most studied virus, only a relatively small group of cellular proteins have been shown essential for HIV-1 propagation. A genetic/proteomic approach has been developed that utilizes a random mutagenic protocol followed by a stringent step of biological selection in efforts to pinpoint those rare sites within the HIV-1 proteome that can accommodate the incorporation of a potent affinity tag without significant loss of those structure/function relationships that are required for viral replication-competency. In conjunction with a novel cryogenic methodology to capture and preserve transient viral-host interactions during immunoisolation, we have recovered several host proteins in association with the replicating virus but previously obscured from conventional scientific investigation. We are reassured that our method is sound since among the cellular proteins recovered and identified using our methodologies are those previously determined by other groups to interact with HIV-1. Furthermore, a straightforward mass spectrometric technique is presented that discriminates with high accuracy between specific and nonspecific proteins in immunoisolated protein complexes. The combined genetic and proteomic method described is a powerful tool set that can be utilized to identify cellular proteins in transient association with a given viral protein at points during the viral life cycle, a subset of which may be absolutely required by the virus for its livelihood but in part, dispensable by the host cell.